Lipase Degradation Resistant Surfactants for Use in Large Molecule Therapeutic Formulations

ABSTRACT

The present invention is directed to pharmaceutical formulations of therapeutic proteins that comprise one or more polyethoxylated fatty alcohol (PFA) surfactants that are resistant to lipase mediated degradation. The present invention is also directed to methods of reducing aggregate and/or particulate formation in pharmaceutical formulations of therapeutic proteins and methods of maintaining a stable surfactant level in pharmaceutical formulations of therapeutic proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 62/721,884, filed 23 Aug. 2018. The entire contents of theaforementioned application is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention is directed to pharmaceutical formulations oftherapeutic proteins that comprise one or more lipase resistantsurfactants. The present invention is also directed to methods ofreducing aggregate and/or particulate formation in pharmaceuticalformulations comprising therapeutic proteins and extending theshelf-life of such pharmaceutical formulations.

BACKGROUND OF THE INVENTION

The inclusion of surfactants in large molecule formulations is acommonly used strategy for stabilizing the biological molecules,preventing adherence to surfaces, avoiding turnover at the air-waterinterface, protecting against surface-induced denaturation, and limitingself-association events that would otherwise lead to aggregation.Protein aggregation can occur during drug processing, long term storage,shipment, and during administration. However, the addition of asurfactant has been shown to minimize interfacial interactions that maystress proteins during filtration, agitation, freeze-thaw,lyophilization, reconstitution, administration, and storage. Currently,the dominant surfactants used by the pharmaceutical industry in largemolecule commercial formulations are polysorbates, e.g., polysorbate 20(PS20) and polysorbate 80 (PS80). Each of these surfactants has theiradvantages, e.g., they decrease protein self-association at an interfaceand prevent protein aggregation. However, a primary disadvantage oftheir use relates to their oxidative degradation (Kerwin B A,“Polysorbates 20 and 80 Used in the Formulation of ProteinBiotherapeutics: Structure and Degradation Pathways,” J. Pharm. Sci.97(8): 2924-35 (2008), which is hereby incorporated by reference in itsentirety) and catalytic degradation (LaBrenz S R, “Ester Hydrolysis ofPolysorbate 80 in mAb Drug Product: Evidence in Support of theHypothesized Risk After the Observation of Visible Particulate in mAbFormulations,” J. Pharm. Sci., 103:2268-2277 (2014), which is herebyincorporated by reference in its entirety), which, in turn, negativelyimpacts the stability and shelf-life of large molecule formulations.

Oxidative degradation of polysorbates can be mitigated in proteinformulations by co-formulating with antioxidants (e.g., methionine) orwith tryptophan (see, e.g., U.S. Patent Application Publication No.2014/0322203 to Alavattam et al.). However, the enzymatic degradation ofpolysorbates, which has been attributed to the presence of host celllipases and esterases (LaBrenz S R, “Ester Hydrolysis of Polysorbate 80in mAb Drug Product: Evidence in Support of the Hypothesized Risk Afterthe Observation of Visible Particulate in mAb Formulations,” J. Pharm.Sci., 103:2268-2277 (2014)), remains a significant challenge tobiopharmaceutical development. While purification processes exist toremove host cell proteins (HCPs), such processes are often inadequate atremoving all HCPs and they are typically cost prohibitive to implementat the manufacturing level. As a result, trace quantities of certainhost cell proteins, including some lipases, are typically retained inbiopharmaceutical products (Chiu et al., “Knockout of aDifficult-to-Remove CHO Host Cell Protein, Lipoprotein Lipase, forImproved Polysorbate Stability in Monoclonal Antibody Formulations,”Biotech. Bioeng. 114(5): 1006-1015 (2017)). Thus, there is a need in theart for the identification of surfactants that possess the advantageousproperties of polysorbate surfactants without some of the disadvantagesrelated to degradation, i.e., effective at preventing proteinaggregation and protein turnover, but resistant to host cell proteinmediated degradation.

The present invention is directed at overcoming the deficiencies in theart related to host cell protein mediated degradation of surfactants inlarge molecule formulations.

SUMMARY OF THE INVENTION

A first aspect of the present invention is directed to a pharmaceuticalformulation. The pharmaceutical formulation of the present inventioncomprises about 20 mg/mL to about 200 mg/mL of a therapeutic protein, apharmaceutically acceptable carrier, and one or more polyethoxylatedfatty alcohol (PFA) surfactants, where the one or more PFA surfactantsis resistant to lipase degradation

Another aspect of the present invention is directed to a method ofreducing aggregate and/or particulate formation in a pharmaceuticalformulation that comprises a biological composition. This methodinvolves providing a biological composition, where the biologicalcomposition comprises about 20 mg/mL to about 200 mg/mL of therapeuticprotein and incorporating one or more lipase resistant polyethoxylatedfatty alcohol (PFA) surfactants in the biological composition as areplacement for a polysorbate surfactant.

Another aspect of the present invention is directed to a method ofextending the shelf life of a pharmaceutical formulation that comprisesa biological composition. This method involves providing a biologicalcomposition, where the biological composition comprises about 20 mg/mLto about 200 mg/mL of therapeutic protein, and incorporating one or morelipase resistant polyethoxylated fatty alcohol (PFA) surfactants in thebiological composition as a replacement for a polysorbate surfactant.

Another aspect of the present invention is directed to a method ofproducing a pharmaceutically acceptable therapeutic protein formulationcomprising a stable surfactant concentration. This method involvesproviding a pharmaceutically acceptable therapeutic protein compositionand incorporating one or more lipase resistant polyethoxylated fattyalcohol (PFA) surfactants in the therapeutic protein composition,thereby producing a pharmaceutically acceptable therapeutic formulationcomprising a surfactant concentration that remains stable over the shelflife of the formulation.

Polysorbates are currently the standard surfactant used by thepharmaceutical industry. However, degradation of polysorbate can occurin large molecule formulations as a result of residual host cell proteinimpurities, namely lipases. The decrease of intact polysorbate levelsover time, together with the formation of polysorbate degradantparticles (e.g., free fatty acids) and large molecule destabilizingpolysorbate degradants, lead to formulation instability, reduced drugproduct shelf life, longer development timelines, and more frequentmanufacturing campaigns, all of which reduce patient access totherapeutic molecules. As demonstrated herein, it was unexpectedly foundthat polyethoxylated fatty alcohol (PFA) surfactants function tostabilize biotherapeutics the same as, and in some cases better than,their counterpart polysorbate surfactants, e.g., polysorbate 20 andpolysorbate 80. Unlike polysorbate surfactants, PFAs are resistant todegradation by host cell lipases; therefore, the concentration of thePFA surfactant remains stable in the formulation overtime and able toprotect the therapeutic molecule of the formulation from aggregation andparticulate formation. As a result, the stability of the therapeuticmolecule formulation is significantly improved as compared to acorresponding therapeutic molecule formulation containing polysorbatesurfactant when the surfactants are exposed to lipases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are graphs showing the protective effect of polysorbatesurfactants (i.e., polysorbate 20 (PS20) and polysorbate 80 (PS80)) andpolyethoxylated fatty alcohol (PFA) surfactants (i.e., Brij® O20 andBrij® L23) on antibody stability after exposure to shaking or thermalstress as measured by size exclusion chromatography (SEC). The graphsshow percent monomer present in each formulation at time zero (T0; whitebar), after shaking stress (72 hours of shaking at 250 RPM at ambienttemperature; grey bars), and after thermal stress (3 month incubation at25° C., followed by shaking stress of 72 h at 250 RPM; black bars).Three different antibody formulations were tested including bispecificmAb A (FIG. 1A), bispecific mAb B (FIG. 1B), and IgG4 mAb C (FIG. 1C).Details of antibody formulations are described herein in the Examples.

FIG. 2 is a graph showing surfactant level (% w/v) in stock solutionsexposed to lipases immobilized on beads over the course of 18 days. Thelevels of intact polysorbate 20 (PS20) and polysorbate 80 (80) declinedsignificantly over the course of exposure, whereas the levels of PFAsurfactants (i.e., Brij® O20 and Brij® L23) remained relativelyconstant.

FIGS. 3A-3C are graphs showing the protective effects of polysorbate andPFA surfactants in three different antibody formulations followinglipase exposure of the surfactants as assessed by SEC. Antibodystability was assessed based on percent monomer present in eachformulation at T0 (white bars), after shaking stress (72 hours ofshaking at 250 RPM at ambient temperature) (grey bars), and afterthermal stress (3 month incubation at 25° C., followed by shaking stressof 72 h at 250 RPM) (black bars). The three antibody formulations thatwere tested include bispecific mAb A (FIG. 3A), bispecific mAb B (FIG.3B), and IgG4 mAb C (FIG. 3C).

FIGS. 4A-4C are graphs showing the protective effects of polysorbate andPFA surfactants in three different antibody formulations followinglipase exposure of the surfactants as assessed by dynamic lightscattering (DLS). DLS analysis was used to measure particle sizedistribution in each antibody formulation after thermal stress (3 monthsat 25° C. and shaking 72 hours at 250 RPM). Particles binning to peak 1(2-8 nm) represent monomers, while particles in peaks 2 to peak 4represent larger particulate ranging from 8 nm to 10,000 nm, which areindicative of aggregates. FIG. 4A shows the DLS results for bispecificmAb A, FIG. 4B shows the DLS results for bispecific mAb B, and FIG. 4Cshows the DLS results for IgG4 mAb C.

FIG. 5 is a graph showing esterase/lipase activity in mAb D formulationswith concentrations of 90 mg/ml and 5 mg/ml. Controls for the assayincluded, NaOH, HCl, porcine esterase (Sigma Product No.: E2884-5KU),water, and formulation buffer without protein.

DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the present invention is directed to a pharmaceuticalformulation. The pharmaceutical formulation of the present inventioncomprises about 20 mg/mL to about 200 mg/mL of a therapeutic protein, apharmaceutically acceptable carrier, and one or more polyethoxylatedfatty alcohol (PFA) surfactants, where the one or more PFA surfactantsis resistant to lipase degradation.

The “pharmaceutical formulation” of the present invention is apreparation which is in a form that permits the biological activity ofthe active ingredient to be effective, and which contains no additionalcomponents that are unacceptably toxic to a subject to which theformulation is administered.

In accordance with this aspect of the present invention, thepharmaceutical formulation is a biopharmaceutical formulation comprisinga therapeutic protein. A “therapeutic protein” as used hereinencompasses any therapeutic product made of two or more coupled aminoacids. Therapeutic proteins include non-recombinant, serum isolatedproteins and peptide or polypeptide fragments thereof, recombinantproteins and peptide or polypeptide fragments thereof, antibodies andantigen binding portions thereof, and antibody mimetics. Therapeuticproteins also include recombinant and non-recombinant fusion proteinsand peptides, chimeric proteins and peptides, and protein and peptideconjugates, and well as antibody and antibody fragment fusions (e.g., Fcfusion protein), chimeras, and conjugates. Therapeutic proteins alsoinclude engineered protein scaffolds, e.g., fibronectin type III domainscaffold binding proteins or monobodies.

In one embodiment, the therapeutic protein is an antibody. Antibodiesinclude both full length antibodies, antigen binding fragments thereof,and antibody derivatives. Suitable antibodies include polyclonal andmonoclonal antibodies of any class (e.g., IgG, IgE, IgM, IgD, and IgA),and any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2). Theantibody can be a humanized antibody, a human antibody, a chimericantibody, a CDR-grafted antibody, a multispecific antibody (e.g.,bi-specific or tri-specific antibodies). Suitable antibody fragmentsinclude, without limitation, any molecule containing an antigen bindingregion or antigen binding domain of a full antibody, e.g., single domainantibodies comprising the heavy chain variable region (V_(H)) or thelight chain variable region (V_(L)). In one embodiment, the antibodyfragment comprises a single-chain polypeptide containing a portion ofthe light-chain variable domain (e.g., one, two or three of thecomplementary determining regions (CDRs)), or a single-chain polypeptidecontaining a portion of the heavy chain variable domain. Other suitableantibody fragments encompassed by the present invention includeantigen-binding (F(ab)) fragments and F(ab′)₂ fragments.

In another embodiment, the therapeutic protein is an antibodyderivative. Antibody derivatives include, for example and withoutlimitation, single-chain antibodies (scFv), tandem scFvs, diabodies,triabodies, or linear antibodies.

Exemplary therapeutic antibodies of the pharmaceutical formulation asdescribed herein include, without limitation, the anti-TNF-α/IL-17Aduobody CNTO 9762 (U.S. Patent Application Publication No. 20170218092to Chiu et al., which is hereby incorporated by reference in itsentirety); the anti-CD38 antibody Daratumumab (U.S. Patent ApplicationPublication No. 20160367663 to Doshi et al., which is herebyincorporated by reference in its entirety); the anti-thrombin antibodyIchorcumab (U.S. Pat. Nos., 9,518,128 and 9,605,082 to Huntington et al,which are hereby incorporated by reference in their entirety); theanti-EGFR/c-Met duobody CNTO 4424 (U.S. Pat. No. 9,695,242 to Chiu etal., which is hereby incorporated by reference in its entirety); theanti-IL23 antibody Guselkumab (CNTO-1959) (U.S. Pat. No. 7,935,344 toBenson et al, which is hereby incorporated by reference in itsentirety); the anti-IL12 antibody Ustekinumab (Stelara®) (U.S. Pat. No.6,902,734 to Giles-Komar et al, which is hereby incorporated byreference in its entirety); erythropoietin (EPO)-mimetic peptideantibody fusion proteins CNTO 528 or CNTO 530 (U.S. Pat. No. 7,241,733to Heavner et al., which is hereby incorporated by reference in itsentirety); the anti-TNFα antibody infliximab (U.S. Pat. No. 5,656,272 toLe et al., which is hereby incorporated by reference in its entirety);and the platelet-specific antibody Abciximab (U.S. Pat. No. 5,770,198 toColler et al., which is hereby incorporated by reference in itsentirety).

The concentration of the therapeutic protein in the pharmaceuticalformulation will vary depending upon numerous factors including, withoutlimitation, the activity of the therapeutic protein, the condition beingtreated, the age of the intended recipient population, route ofadministration, among other things. Thus, the pharmaceutical formulationof the present invention comprises a concentration of a therapeuticprotein in the range from about 10 mg/mL to about 250 mg/mL or any rangebetween these values. In some embodiments, the therapeutic protein is ata concentration greater than about 250 mg/mL. In some embodiments, thetherapeutic protein is at a concentration in the range from any one ofabout 10 mg/mL to 250 mg/mL, 50 mg/mL to 250 mg/mL, 100 mg/mL to 250mg/mL, 150 mg/mL to 250 mg/mL, 200 mg/mL to 250 mg/mL, 10 mg/mL to 200mg/mL, 20 mg/mL to 200 mg/mL, 50 mg/mL to 200 mg/mL, 100 mg/mL to 200mg/mL, 150 mg/mL to 200 mg/mL, 10 mg/mL to 150 mg/mL, 50 mg/mL to 150mg/mL, 100 mg/mL to 150 mg/mL, 10 mg/mL to 100 mg/mL, 50 mg/mL to 100mg/mL, 10 mg/mL to 50 mg/mL or any range between these ranges.

In accordance with this aspect of the present invention, the therapeuticprotein of the pharmaceutical formulation is one that is produced from abiological source, e.g., a primary population of cells or animmortalized cell line. In one embodiment, the therapeutic protein isproduced in a mammalian cell line. Suitable mammalian cell lines includehamster cell lines such as Chinese Hamster Ovary (CHO) cell linesCHODG44 and DUXB11 (Gibco, Gaithersburg, Md.), CHO-K1 (American TypeCulture Collection (ATCC) #CCL-61), CHO-S (Gibco), Freestyle CHO-S(Invitrogen, Carlsbad, Calif.),CHO-T (Acyte, Brisbane, Australia), andCHO3E7 (National Research Council of Canada (CNRC) # L-11992). Suitablemammalian cell lines also include mouse cell lines, e.g., the mousemyeloma NS0 cell line (European Collection of Authenticated CellCultures (ECACC) #85110503) and mouse myeloma cell line Sp2/0(ATCC-CRL-1581). Suitable mammalian cells also include human cell linessuch as human amniocyte, e.g., CAP cells and CAP-T cells (Cevec, Koln,Germany), human retina cells, e.g., PER.C6 cells (Crucell, Leiden,Netherlands), or human embryonic kidney cells, e.g., Freestyle HEK2930Fcells (Invitrogen), HEK 293 6E (CNRC #L-11266), HEK 293 T (ATCC#CRL-11268).

The lipase or esterase activity in the pharmaceutical formulation asdescribed herein will vary depending upon numerous factors including,without limitation, the concentration of the therapeutic protein, thebiological source of the therapeutic protein, and methods of therapeuticprotein purification employed. In one embodiment, the lipase activity inthe pharmaceutical formulation is >1 unit/mL of purified porcineesterase or equivalent thereof, where one “unit” of lipase/esteraseactivity hydrolyzes 1.0 μmole of ethyl butyrate to butyric acid andethanol per minute at pH 8.0 at 25° C.

The pharmaceutical formulation of the present invention also comprisesone or more polyethoxylated fatty alcohol (PFA) surfactants. Theincorporation of one or more PFA surfactants is particularlyadvantageous in pharmaceutical formulations containing a therapeuticprotein that was produced in a biological system, like a cell, becausePFAs are resistant to degradation by residual host cell proteins thatare carried over to the formulation. In particular, PFAs are resistantto host cell lipases that cannot be adequately removed from thetherapeutic protein preparation, and which are highly active at very lowconcentrations.

PFAs (also referred to as alcohol ethoxylates) encompass a class ofnon-ionic surfactants that contain a hydrophobic alkyl chain attachedvia an ether linkage to a hydrophilic ethylene oxide (EO) chain. Thisclass of surfactants is defined by a general structure of Formula I.

R(OCH₂CH₂)_(n)OH   Formula I

The alkyl chain, R, of the PFA varies in length and degree of linearity,but is typically between 8 and 18 carbons in length, in someembodiments, between 11-15 carbons in length. The length of the EO chain(i.e., n of formula I) also varies in length from about 1 to about 40 EOunits. In one embodiment, the PFA of the pharmaceutical formulation ofthe present invention comprises a PFA having an ethylene oxide chaincomprising about 5 to about 40 ethylene oxide units (i.e., 5, 6, 7, 8,9, 10, 11, 12 ,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 38, 39, or 40 EO units).

While PFAs are surfactants that are used primarily in laundry detergentsand other household products, PFAs have not been used in pharmaceuticalformulations of biologic molecules, in particular, pharmaceuticalformulations comprising therapeutic proteins, such as antibodies.However, as demonstrated herein, it was unexpectedly found, that thisclass of surfactants function the same as, and in some cases better thanpolysorbate surfactants, e.g., polysorbate 20 and polysorbate 80, whichare the predominant surfactants utilized by the pharmaceutical industryin large molecule commercial formulations. Unlike polysorbatesurfactants, PFAs are resistant to degradation by host cell lipases;therefore, the concentration of the PFA surfactant remains stable in theformulation overtime and able to protect the therapeutic protein of theformulation from aggregation and particulate formation. As a result, thestability of the therapeutic protein formulation is significantlyimproved as compared to a corresponding therapeutic protein formulationcontaining polysorbate surfactant when the surfactants are exposed tolipases.

In one embodiment, the pharmaceutical formulation of the presentinvention comprises a polyoxyethylene lauryl ether (CAS No. 9002-92-0).Exemplary polyoxyethylene lauryl ethers include, without limitation,polyoxyethylene (23) lauryl ether (also known by the tradenames Brij®L23 and Brij® 35); polyoxyethylene (4) lauryl ether (also known aspolyethylene glycol dodecyl ether and Brij® L4); and polyoxyethylene(10) lauryl ether (also known as decaethylene glycol monododecyl ether).

In another embodiment, the pharmaceutical formulation of the presentinvention comprises a polyoxyethylene oleyl ether (CAS No. 9004-98-2).Exemplary polyoxyethylene oleyl ethers include, without limitation,polyoxyethylene (20) oleyl ether (also know by the tradenames Brij® 98,Brij® 99, and Brij® O20); polyoxyethylene (10) oleyl ether (also know bythe tradenames Brij® O10 and Brij® 97); and polyoxyethylene (2) oleylether (also know by the tradenames Brij® 93 and polyethylene glycololeyl ether).

In another embodiment, the pharmaceutical formulation of the presentinvention comprises a polyoxyethylene stearyl ether (Cas No. 9005-00-9).Exemplary polyoxyethylene stearyl ethers include, without limitation,polyoxyethylene (20) stearyl ether (also know by the tradename Brij®S20); polyoxyethylene (100) stearyl ether (also known by the tradenameBrij® S100); polyoxyethylene (10) stearyl ether (also known by thetradenames Brij® S10 and polyethylene glycol octadecyl ether); andpolyoxyethylene (2) stearyl ether (also known by the tradename Brij®S2).

In another embodiment, the pharmaceutical formulation of the presentinvention comprises a polyoxyethylene cetyl ether (Cas No. 9004-95-9).Exemplary polyoxyethylene cetyl ethers include, without limitation,polyoxyethylene (20) cetyl ether (also known by the tradenames Brij® 58and polyethylene glycol hexadecyl ether); polyoxyethylene (2) cetylether (also known by the tradename Brij® 52); and polyoxyethylene (10)cetyl ether (also known by the tradename Brij® C10).

In one embodiment, the pharmaceutical formulation of the presentinvention comprises about 0.001% to about 0.4% (w/v) of PFA surfactant.In one embodiment, the pharmaceutical formulation comprises about 0.005%to about 0.2% (w/v) of the PFA surfactant. In one embodiment, thepharmaceutical formulation comprises about 0.01% to about 0.1% (w/v) ofPFA surfactant (i.e., about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%,0.07%, 0.08%, 0.09%, 0.1%). In another embodiment, the pharmaceuticalformulation comprises about 0.01% to about 0.09% (w/v) of PFAsurfactant. In another embodiment, the pharmaceutical formulationcomprises about 0.01% to about 0.06% (w/v) of PFA surfactant. In anotherembodiment, the pharmaceutical formulation comprises about 0.01% toabout 0.04% (w/v) of PFA surfactant.

The pharmaceutical formulation of the present invention is a stableformulation. As referred to herein, a “stable formulation” is one inwhich the protein therein essentially retains its physical stabilityand/or chemical stability and/or biological activity upon storage.Preferably, the formulation essentially retains its physical andchemical stability, as well as its biological activity upon storage.Steady levels of PFA surfactant during processing and storage contributeto the stability of the formulations describe herein. In one embodiment,the formulation retains 70% of its starting level of PFA underappropriate storage conditions. In another embodiment, the formulationretains 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% of its starting level of PFA surfactant under appropriatestorage conditions.

In some embodiments, >90% of the PFA remains intact after theformulation is stored at about 1° C. to about 10° C. for about sixmonths, at least about 12 months, at least about 18 months, at leastabout 24 months, at least about 30 months, at least about 36 months, atleast about 42 months, or at least about 48 months.

In some embodiments, >95% of the PFA remains intact in the formulationafter the formulation is stored at about 1° C. to about 10° C. for atleast about six months, at least about 12 months, at least about 18months, at least about 24 months, at least about 30 months, at leastabout 36 months, at least about 42 months, or at least about 48 months.

In another embodiment, >90% of the PFA remains intact after theformulation is stored at about 22° C. to about 28° C. for at least aboutone month, at least about two months, at least about three months, atleast about four months, at least about five months, at least about sixmonths, at least about seven months, at least about eight months, atleast about nine months, at least about ten months, at least abouteleven months, or at least about twelve months. In someembodiments, >95% of the PFA remains intact after the formulation isstored at about 22° C. to about 28° C. for at least about one month, atleast about two months, at least about three months, at least about fourmonths, at least about five months, at least about six months, at leastabout seven months, at least about eight months, at least about ninemonths, at least about ten months, at least about eleven months, or atleast about twelve months.

In another embodiment, >90% of the PFA remains intact after theformulation is stored at about −15° C. to about −90° C. for at leastabout 12 months, at least about 18 months, at least about 24 months, atleast about 30 months, at least about 36 months, at least about 42months, at least about 48 months, at least about 54 months, at leastabout 60 months, at least about 66 months, or at least about 72 months.In some embodiments, >95% of the PFA remains intact after theformulation is stored at about −15° C. to about −90° C. for at leastabout 12 months, at least about 18 months, at least about 24 months, atleast about 30 months, at least about 36 months, at least about 42months, at least about 48 months, at least about 54 months, at leastabout 60 months, at least about 66 months, or at least about 72 months.

Various analytical techniques known in the art can be utilized formeasuring protein stability in the pharmaceutical formulations of thepresent invention. Stability can be measured at a selected amount oflight exposure and/or temperature for a selected time period. Stabilitycan be evaluated qualitatively and/or quantitatively in a variety ofdifferent ways, including evaluation of aggregate formation. Aggregateformation can be evaluated by, for example and without limitation, sizeexclusion chromatography, turbidity, and/or by visual inspection.Stability can also be evaluated based on particulate and sub-visibleparticulate formation, which can be assessed using, for example andwithout limitation, dynamic light scattering, nanoparticle trackinganalysis, resonant mass measurement, light obscuration, and flowimaging. In another embodiment, stability is measured by evaluation ofROS formation, using for example and without limitation, a light stressassay or a 2,2′-Azobis(2-Amidinopropane) Dihydrochloride (AAPH) stressassay. In another embodiment, stability is assessed based on theoxidation of specific amino acid residues of the protein. This analysiscan be carried out using antibody detection. For example, detection of aTrp residue and/or a Met residue by monoclonal antibody detection. Inanother embodiment, stability is evaluated by assessing chargeheterogeneity using cation exchange chromatography, image capillaryisoelectric focusing (icIEF) or capillary zone electrophoresis. Othermeasures of stability that are suitable for use in accordance with themethods described herein include, without limitation, amino-terminal orcarboxy-terminal sequence analysis; mass spectrometric analysis;SDS-PAGE or capillary electrophoresis SDS analysis to compare reducedand intact antibody; peptide map (for example tryptic or LYS-C)analysis; and evaluating biological activity or target binding functionof the protein (e.g., antigen binding function of an antibody.

In one embodiment the pharmaceutical formulation of the presentinvention has enhanced stability as compared to pharmaceuticalformulations comprising the same therapeutic protein but formulated witha polysorbate surfactant. In one embodiment, the stability of thepharmaceutical formulation of the present invention is characterized bythe percentage of therapeutic protein in the formulation that maintainsits desired monomeric state (vs. a dimer or trimer aggregated state).Accordingly, in one embodiment, >90% of the therapeutic protein in thepharmaceutical formulation comprising a PFA surfactant as describedherein is in a monomeric state for its shelf-life. In anotherembodiment, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of thetherapeutic protein in the pharmaceutical formulation is in the desiredmonomeric state for the entirety of its shelf-life.

In another embodiment, >90% of the therapeutic protein is in a monomericstate after the formulation is stored at about 22° C. to about 28° C.for at least about one month, at least about two months, at least aboutthree months, at least about four months, at least about five months, atleast about six months, at least about seven months, at least abouteight months, at least about nine months, at least about ten months, atleast about eleven months, or at least about twelve months. In anotherembodiment, >95% of the therapeutic protein is in a monomeric stateafter the formulation is stored at about 22° C. to about 28° C. for atleast about one month, at least about two months, at least about threemonths, at least about four months, at least about five months, at leastabout six months, at least about seven months, at least about eightmonths, at least about nine months, at least about ten months, at leastabout eleven months, or at least about twelve months.

In another embodiment, >90% of the therapeutic protein is in a monomericstate after the formulation is stored at about 1° C. to about 10° C. forabout six months, at least about 12 months, at least about 18 months, atleast about 24 months, at least about 30 months, at least about 36months, at least about 42 months, or at least about 48 months. In someembodiments, >95% of the therapeutic protein is in a monomeric stateafter the formulation is stored at about 1° C. to about 10° C. for atleast about six months, at least about 12 months, at least about 18months, at least about 24 months, at least about 30 months, at leastabout 36 months, at least about 42 months, or at least about 48 months.

In another embodiment, >90% of the therapeutic protein is in a monomericstate after the formulation is stored at about −15° C. to about −90° C.for at least about 12 months, at least about 18 months, at least about24 months, at least about 30 months, at least about 36 months, at leastabout 42 months, at least about 48 months, at least about 54 months, atleast about 60 months, at least about 66 months, or at least about 72months. In another embodiment, >95% of the therapeutic protein is in amonomeric state after the formulation is stored at about −15° C. toabout −90° C. for at least about 12 months, at least about 18 months, atleast about 24 months, at least about 30 months, at least about 36months, at least about 42 months, at least about 48 months, at leastabout 54 months, at least about 60 months, at least about 66 months, orat least about 72 months.

In another embodiment, the formulation of the present invention is astable formulation, as shown by its resistance to sub-visibleparticulate formation. In one embodiment, the formulations as describedherein contain ≤6000 particles (≥10 μm in size) per container (e.g., 1mL to 50 mL vial or 0.25 mL to 2 mL syringe) and ≤600 particles (≥25 μmin size) per container after storage at about 22° C. to about 28° C. forat least about one month, at least about two months, at least aboutthree months, at least about four months, at least about five months, atleast about six months, at least about seven months, at least abouteight months, at least about nine months, at least about ten months, atleast about eleven months, or at least about twelve months.

In another embodiment, the therapeutic protein pharmaceuticalformulations described herein are resistance to sub-visible particulateformation during cold storage. In one embodiment, the formulations asdescribed herein contain ≤6000 particles (≥10 μm in size) per containerand ≤600 particles (≥25 μm in size) per container after storage at about1° C. to about 10° C. for about six months, at least about 12 months, atleast about 18 months, at least about 24 months, at least about 30months, at least about 36 months, at least about 42 months, or at leastabout 48 months.

In another embodiment, the therapeutic protein pharmaceuticalformulations described herein are resistance to sub-visible particulateformation during long-term cold storage. In one embodiment, theformulations as described herein contain ≤6000 particles (≥10 μm insize) per container and ≤600 particles (≥25 μm in size) per containerafter storage at about −15° C. to about −90° C. for at least about 12months, at least about 18 months, at least about 24 months, at leastabout 30 months, at least about 36 months, at least about 42 months, atleast about 48 months, at least about 54 months, at least about 60months, at least about 66 months, or at least about 72 months.

In another embodiment, the therapeutic protein pharmaceuticalformulations described herein are stable formulations as demonstrated bytheir resistance to visible particulate formation. In one embodiment,the formulations as described herein contain ≤80 particles (≥70 μm insize) per milliliter after storage at about 22° C. to about 28° C. forat least about one month, at least about two months, at least aboutthree months, at least about four months, at least about five months, atleast about six months, at least about seven months, at least abouteight months, at least about nine months, at least about ten months, atleast about eleven months, or at least about twelve months. In anotherembodiment, the formulations as described herein contain ≤10 particles(≥70 μm in size) per milliliter after storage at about 22° C. to about28° C. for at least about one month, at least about two months, at leastabout three months, at least about four months, at least about fivemonths, at least about six months, at least about seven months, at leastabout eight months, at least about nine months, at least about tenmonths, at least about eleven months, or at least about twelve months.

The stable therapeutic protein pharmaceutical formulations as describedherein are resistant to visible particulate formation during coldstorage. In one embodiment, the formulations as described herein contain≤80 particles (≥70 μm in size) per milliliter after storage at about 1°C. to about 10° C. for about six months, at least about 12 months, atleast about 18 months, at least about 24 months, at least about 30months, at least about 36 months, at least about 42 months, or at leastabout 48 months. In another embodiment, the formulations as describedherein contain ≤10 particles (≥70 μm in size) per milliliter afterstorage at about 1° C. to about 10° C. for at least about six months, atleast about 12 months, at least about 18 months, at least about 24months, at least about 30 months, at least about 36 months, at leastabout 42 months, or at least about 48 months.

In another embodiment, the therapeutic protein pharmaceuticalformulations described herein are resistant to visible particulateformation during long-term cold storage. In one embodiment, theformulations as described herein contain ≤80 particles (≥70 μm in size)per milliliter after storage at about −15° C. to about −90° C. for atleast about 12 months, at least about 18 months, at least about 24months, at least about 30 months, at least about 36 months, at leastabout 42 months, at least about 48 months, at least about 54 months, atleast about 60 months, at least about 66 months, or at least about 72months. In another embodiment, the formulations as described hereincontain ≤10 particles (≥70 μm in size) per milliliter after storage atabout −15° C. to about −90° C. for at least about 12 months, at leastabout 18 months, at least about 24 months, at least about 30 months, atleast about 36 months, at least about 42 months, at least about 48months, at least about 54 months, at least about 60 months, at leastabout 66 months, or at least about 72 months.

In another embodiment, the stable formulations of the present inventionhave low turbidity. In one embodiment, the turbidity of the formulationsas described herein is ≤18.0 NTU (Nephelometric Turbidity Unit) afterstorage at about 22° C. to about 28° C. for at least about one month, atleast about two months, at least about three months, at least about fourmonths, at least about five months, at least about six months, at leastabout seven months, at least about eight months, at least about ninemonths, at least about ten months, at least about eleven months, or atleast about twelve months.

In another embodiment, the stable therapeutic protein pharmaceuticalformulations have low turbidity during cold storage. In one embodiment,the turbidity of the formulations as described herein is ≤18.0 NTU afterstorage at about 1° C. to about 10° C. for about six months, at leastabout 12 months, at least about 18 months, at least about 24 months, atleast about 30 months, at least about 36 months, at least about 42months, or at least about 48 months.

In another embodiment, the stable therapeutic protein pharmaceuticalformulations have low turbidity during cold storage. In one embodiment,the turbidity of the formulations as described herein is ≤18.0 NTU afterstorage at about −15° C. to about −90° C. for at least about 12 months,at least about 18 months, at least about 24 months, at least about 30months, at least about 36 months, at least about 42 months, at leastabout 48 months, at least about 54 months, at least about 60 months, atleast about 66 months, or at least about 72 months.

The pharmaceutical formulation of the present invention can furthercomprise at least one of any suitable auxiliary agents, such as, but notlimited to, diluents, binders, stabilizers, buffers, salts, lipophilicsolvents, preservatives, adjuvants or the like. Non-limiting examplesof, and methods of preparing such sterile solutions are well known inthe art, see, e.g., Gennaro A R., Remington's Pharmaceutical Sciences,18^(th) ed, Mack Publishing Co. (1990), which is hereby incorporated byreference in its entirety.

Pharmaceutical excipients and additives useful in the presentcomposition include, but are not limited to, proteins, peptides, aminoacids, lipids, and carbohydrates (e.g., sugars, includingmonosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatizedsugars, such as alditols, aldonic acids, esterified sugars and the like;and polysaccharides or sugar polymers), which can be present singly orin combination, comprising alone or in combination 1-99.99% by weight orvolume. Exemplary protein excipients include serum albumin, such ashuman serum albumin (HSA), recombinant human albumin (rHA), gelatin,casein, and the like. Representative amino acid/antibody components,which can also function in a buffering capacity, include alanine,glycine, arginine, betaine, histidine, glutamic acid, aspartic acid,cysteine, lysine, leucine, isoleucine, valine, methionine,phenylalanine, aspartame, and the like.

Carbohydrate excipients suitable for use in the invention include, forexample, monosaccharides, such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol,sorbitol (glucitol), myoinositol and the like. The carbohydrateexcipient comprises from about 0.5% to about 15% w/v of thepharmaceutical formulation described herein.

The formulation of the present invention can also include a buffer or apH-adjusting agent. Typically, the buffer is a salt prepared from anorganic acid or base. Representative buffers include organic acid salts,such as salts of citric acid, ascorbic acid, gluconic acid, carbonicacid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris,tromethamine hydrochloride, or phosphate buffers. The buffering saltsare typically present in pharmaceutical formulations of the presentinvention at a concentration of about 5 mM to about 50 mM.

The pH of the formulations can range from about pH 4 to about pH 10,from about pH 5 to about pH 9, or from about pH 6 to about pH 8. In oneembodiment, the formulations of the present invention have a pH betweenabout pH 6.8 and about pH 7.8. Preferred buffers include phosphatebuffers and sodium phosphate buffers, e.g., phosphate buffered saline(PBS).

Additionally, the pharmaceutical formulation of the present inventioncan include polymeric excipients/additives, such aspolyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g.,cyclodextrins, such as 2-hydroxypropyl-β-cyclodextrin), polyethyleneglycols, flavoring agents, antimicrobial agents, sweeteners,antioxidants, antistatic agents, lipids (e.g., phospholipids, fattyacids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).

The pharmaceutical formulation of the present invention may furthercomprise one or more pharmaceutically acceptable carriers. A“pharmaceutically acceptable carrier” refers to a diluent, adjuvant,excipient, or vehicle with which the therapeutic protein is formulatedwith. Such vehicles may be liquids, such as water and oils, includingthose of petroleum, animal, vegetable or synthetic origin, such aspeanut oil, soybean oil, mineral oil, sesame oil and the like. Forexample, 0.4% saline and 0.3% glycine may be used. These solutions aresterile and generally free of particulate matter. They may be sterilizedby conventional, well-known sterilization techniques (e.g., filtration).

The pharmaceutical formulation described herein may also containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, stabilizing, thickening, lubricating and coloring agents, etc.The concentration of the therapeutic protein in such pharmaceuticalformulations may vary, from less than about 0.5%, usually to at leastabout 1%, to as much as 15%, 20%, 25%, 30%, or >30% by weight and may beselected primarily based on required dose, fluid volumes, viscosities,etc., according to the particular mode of administration selected.Suitable vehicles and formulations, inclusive of other human proteins,e.g., human serum albumin, are described, for example, in REMINGTON: THESCIENCE AND PRACTICE OF PHARMACY, 21^(st) Edition, (2006), which ishereby incorporated by reference in its entirety.

Another aspect of the present invention is directed to a method ofreducing aggregate and/or particulate formation in a pharmaceuticalformulation that comprises a biological composition. This methodinvolves providing a biological composition, where the biologicalcomposition comprises about 20 mg/mL to about 200 mg/mL of therapeuticprotein, and incorporating one or more lipase resistant polyethoxylatedfatty alcohol (PFA) surfactants in the biological composition as areplacement for a polysorbate surfactant.

Another aspect of the present invention is directed to a method ofextending the shelf life of a pharmaceutical formulation that comprisesa biological composition. This method involves providing a biologicalcomposition, where the biological composition comprises about 20 mg/mLto about 200 mg/mL of therapeutic protein, and incorporating one or morelipase resistant polyethoxylated fatty alcohol (PFA) surfactants in thebiological composition as a replacement for a polysorbate surfactant.

Another aspect of the present invention is directed to a method ofproducing a pharmaceutically acceptable therapeutic protein formulationcomprising a stable surfactant concentration. This method involvesproviding a pharmaceutically acceptable therapeutic protein compositionand incorporating one or more lipase resistant polyethoxylated fattyalcohol (PFA) surfactants in the therapeutic protein compositionthereby, producing a pharmaceutically acceptable therapeutic formulationcomprising a surfactant concentration that remains stable over the shelflife of the formulation.

The methods as described herein are particularly suitable for biologicalcompositions having a lipase/esterase activity that is ≥1 unit/mL ofpurified porcine esterase or equivalent thereof, where one “unit” ofactivity hydrolyzes 1.0 μmole of ethyl butyrate to butyric acid andethanol per minute at pH 8.0 at 25° C. It is this level of lipaseactivity in a biological composition that is sufficient to causepolysorbate degradation, subsequently jeopardizing the stability of thebiological composition. Thus, in one embodiment, the methods asdescribed herein involve providing a pharmaceutical formulation of abiological composition, where the biological composition has alipase/esterase activity of ≥1 units/mL of purified porcine esterase orequivalent thereof.

Incorporating the one or more lipase resistance PFA surfactants intothis biological composition as a replacement for a polysorbatesurfactant will mitigate surfactant degradation over time and reduceaggregate and/or particulate formation in the biological composition andin the pharmaceutical formulation. Such pharmaceutical formulations willhave an extended shelf-life and a stable surfactant over the course ofthe formulation's shelf-life. In certain embodiments, the methods asdescribed herein provide a pharmaceutical formulation of a biologicalcomposition with one or more lipase resistance PFA surfactants, whereinthe biological composition has a lipase/esterase activity of ≥1 units/mLof purified porcine esterase or equivalent thereof. In certain otherembodiments, the methods as described herein provide a pharmaceuticalformulation of a biological composition with one or more lipaseresistance PFA surfactants, wherein the biological composition has alipase/esterase activity of ≥1, ≥0.9, ≥0.8, ≥0.7, ≥0.6, ≥0.5, ≥0.4,≥0.3, ≥0.2, ≥0.1 units/mL of purified porcine esterase or equivalentthereof. In certain embodiments, the methods as described herein providea pharmaceutical formulation of a biological composition with one ormore lipase resistance PFA surfactants, wherein the biologicalcomposition has a lipase/esterase activity of ≥0.1 units/mL of purifiedporcine esterase or equivalent thereof.

In another embodiment, the methods of the present invention furtherinvolve measuring lipase activity of the biological composition prior toincorporating the one or more lipase resistant PFA surfactants. Suitablemethods of measuring lipase activity in a biological sample are known inthe art, see, e.g., Hernandez-Garcia et al., “An Improved Method toMeasure Lipase Activity in Aqueous Media,” Anal. Biochem. 530:104-106(2017), Tietz and Repique, “Proposed Standard Method for MeasuringLipase Activity in Serum by a Continuous Sampling Technique,” Clin.Chem. 19(11):1268-1275 (1973), Ehnholm et al., “Two Methods Compared forMeasuring Lipase Activity in Plasma after Heparin Administration,” Clin.Chem. 30(9): 1568-70 (1984), which are hereby incorporated by referencein their entirety. Generally, a suitable method of measuring lipaseactivity of the biological sample involves measuring the conversion of4-nitrophenyl acetate to 4-nitrophenol by the esterase/lipase present inthe sample. This conversion can be monitored, detected, and quantifiedusing spectrophotometric methods. For example, at pH>6.0 the conversionthe 4-nitrophenol is colored yellow and its production can be monitoredat 400 nm. As noted above, a lipase resistant PFA surfactant isincorporated into biological compositions comprising a lipase/esteraseactivity of ≥1 unit/mL of purified porcine esterase or equivalentthereof.

The methods of the present invention can be employed on pharmaceuticalcompositions comprising any of the therapeutic proteins described supra,including, without limitation, non-recombinant, serum isolated proteinsand peptides thereof, recombinant proteins or peptides thereof, anantibody or an antigen binding portion thereof, antibody derivatives,and/or antibody-drug conjugate.

In accordance with these methods of the present invention, thepharmaceutical formulation comprises a therapeutic protein that isderived from a biological source, i.e., a population of cells or a cellline. Cell lines from which the therapeutic protein may be obtained frominclude, without limitation, mammalian CHO cell lines, PER.C6 celllines, and Sp2/0 cell lines as described supra. The concentration of thetherapeutic protein in the formulation ranges from about 10 mg/mL toabout 250 mg/mL or any range between these values as described supra. Insome embodiments, the therapeutic protein is at a concentration greaterthan about 250 mg/mL. In some embodiments, the therapeutic protein is ata concentration in the range from any one of about 10 mg/mL to 250mg/mL, 50 mg/mL to 250 mg/mL, 100 mg/mL to 250 mg/mL, 150 mg/mL to 250mg/mL, 200 mg/mL to 250 mg/mL, 10 mg/mL to 200 mg/mL, 50 mg/mL to 200mg/mL, 100 mg/mL to 200 mg/mL, 150 mg/mL to 200 mg/mL, 10 mg/mL to 150mg/mL, 50 mg/mL to 150 mg/mL, 100 mg/mL to 150 mg/mL, 10 mg/mL to 100mg/mL, 50 mg/mL to 100 mg/mL, 10 mg/mL to 50 mg/mL or any range betweenthese ranges.

In accordance with this aspect of the present invention, theincorporation of one or more PFA surfactants in a pharmaceuticalformulation containing a therapeutic protein that was produced in abiological system, like a cell, is particularly advantageous, becausePFAs are resistant to degradation by residual host cell proteins thatare carried over to the formulation. In particular, PFAs are resistantto host cell lipases that are not, and cannot be, adequately removedfrom the therapeutic protein preparation, and which are highly active atvery low concentrations (≥1 unit/mL of purified porcine esterase orequivalent thereof). As described supra, suitable PFA surfactants havethe general structure of Formula I. In one embodiment, the length of thehydrophilic ethylene oxide chain comprises about 1 to about 40 units. Inone embodiment, the length of the ethylene oxide chain comprises about 5to about 40 ethylene oxide units.

In one embodiment, a polyoxyethylene lauryl ether, e.g., polyoxyethylene(23) lauryl ether (also known by the tradenames Brij® L23 and Brij® 35),is incorporated into the pharmaceutical formulation to reduce aggregateand/or particulate formation in the formulation. In another embodiment,a polyoxyethylene oleyl ether, e.g., polyoxyethylene (20) oleyl ether(also know by the tradenames Brij® 98 and Brij® 99) or polyoxyethylene(10) oleyl ether (also know by the tradenames Brij® O10 and Brij® 97),is incorporated into the pharmaceutical formulation to reduce aggregateand/or particulate formation in the formulation. In another embodiment,a polyoxyethylene cetyl ether, e.g., polyoxyethylene (20) cetyl ether(also known by the tradename Brij® 58), is incorporated into thepharmaceutical formulation to reduce aggregate and/or particulateformation in the formulation. In another embodiment, a polyoxyethylenestearyl ether, e.g., polyoxyethylene (20) stearyl ether (also know bythe tradename Brij® S20), is incorporated into the pharmaceuticalformulation to reduce aggregate and/or particulate formation in theformulation.

In one embodiment, the one or more PFA surfactants are incorporated intothe pharmaceutical formulation such that the composition comprises0.001% to about 0.4% (w/v) of PFA surfactant. In one embodiment, themethod produces a pharmaceutical formulation comprising about 0.005% toabout 0.2% (w/v) of PFA surfactant. In one embodiment, the methodproduces a pharmaceutical formulation comprising about 0.01% to about0.1% (w/v) of PFA surfactant. In another embodiment, the method producesa pharmaceutical formulation comprising about 0.01% to about 0.09% (w/v)of PFA surfactant. In another embodiment, the method produces apharmaceutical formulation comprising about 0.01% to about 0.06% (w/v)of PFA surfactant. In another embodiment, the method produces apharmaceutical formulation comprising about 0.01% to about 0.04% (w/v)of PFA surfactant.

The methods of the present invention produce therapeutic proteinpharmaceutical formulations having a stable surfactant concentration. Inaccordance with these methods, >90% of the PFA remains intact in theformulation after the formulation is stored at about 1° C. to about 10°C. for about six months, at least about 12 months, at least about 18months, at least about 24 months, at least about 30 months, at leastabout 36 months, at least about 42 months, or at least about 48 months.In another embodiment, the methods of the present invention produceformulations where >95% of the PFA remains intact in the formulationafter the formulation is stored at about 1° C. to about 10° C. for atleast about six months, at least about 12 months, at least about 18months, at least about 24 months, at least about 30 months, at leastabout 36 months, at least about 42 months, or at least about 48 months.

In another embodiment, the methods of the present invention produceformulations where >90% of the PFA remains intact after the formulationis stored at about 22° C. to about 28° C. for at least about one month,at least about two months, at least about three months, at least aboutfour months, at least about five months, at least about six months, atleast about seven months, at least about eight months, at least aboutnine months, at least about ten months, at least about eleven months, orat least about twelve months. In another embodiment, the methods of thepresent invention produce formulations where >95% of the PFA remainsintact after the formulation is stored at about 22° C. to about 28° C.for at least about one month, at least about two months, at least aboutthree months, at least about four months, at least about five months, atleast about six months, at least about seven months, at least abouteight months, at least about nine months, at least about ten months, atleast about eleven months, or at least about twelve months.

In another embodiment, the methods of the present invention produceformulations where >90% of the PFA remains intact after the formulationis stored at about −15° C. to about −90° C. for at least about 12months, at least about 18 months, at least about 24 months, at leastabout 30 months, at least about 36 months, at least about 42 months, atleast about 48 months, at least about 54 months, at least about 60months, at least about 66 months, or at least about 72 months. Inanother embodiment, the methods of the present invention produceformulations where >95% of the PFA remains intact after the formulationis stored at about −15° C. to about −90° C. for at least about 12months, at least about 18 months, at least about 24 months, at leastabout 30 months, at least about 36 months, at least about 42 months, atleast about 48 months, at least about 54 months, at least about 60months, at least about 66 months, or at least about 72 months.

In another embodiment, the methods of the present invention producestable therapeutic protein formulations where >90% of the therapeuticprotein (e.g., antibody) is in a monomeric state after the formulationis stored at about 22° C. to about 28° C. for at least about one month,at least about two months, at least about three months, at least aboutfour months, at least about five months, at least about six months, atleast about seven months, at least about eight months, at least aboutnine months, at least about ten months, at least about eleven months, orat least about twelve months. In another embodiment, the methods of thepresent invention produce formulations where >95% of the therapeuticprotein in the formulation is in a monomeric state after the formulationis stored at about 22° C. to about 28° C. for at least about one month,at least about two months, at least about three months, at least aboutfour months, at least about five months, at least about six months, atleast about seven months, at least about eight months, at least aboutnine months, at least about ten months, at least about eleven months, orat least about twelve months.

The methods of the present invention produce stable therapeutic proteinpharmaceutical formulations where >90% of the therapeutic protein (e.g.,antibody) is in a monomeric state after the formulation is stored atabout 1° C. to about 10° C. for about six months, at least about 12months, at least about 18 months, at least about 24 months, at leastabout 30 months, at least about 36 months, at least about 42 months, orat least about 48 months. In some embodiments, the methods describedherein produce therapeutic protein formulations where >95% of thetherapeutic protein is in a monomeric state after the formulation isstored at about 1° C. to about 10° C. for at least about six months, atleast about 12 months, at least about 18 months, at least about 24months, at least about 30 months, at least about 36 months, at leastabout 42 months, or at least about 48 months.

In another embodiment, the methods of the present invention produceformulations where >90% of the therapeutic protein (e.g., antibody) isin a monomeric state after the formulation is stored at about −15° C. toabout −90° C. for at least about 12 months, at least about 18 months, atleast about 24 months, at least about 30 months, at least about 36months, at least about 42 months, at least about 48 months, at leastabout 54 months, at least about 60 months, at least about 66 months, orat least about 72 months. In another embodiment, the methods of thepresent invention produce formulations where >95% of the therapeuticprotein is in a monomeric state after the formulation is stored at about−15° C. to about −90° C. for at least about 12 months, at least about 18months, at least about 24 months, at least about 30 months, at leastabout 36 months, at least about 42 months, at least about 48 months, atleast about 54 months, at least about 60 months, at least about 66months, or at least about 72 months.

In another embodiment, the methods of the present invention producestable formulations resistant to sub-visible particulate formation. Inone embodiment, the formulations as described herein contain ≤6000particles (≥10 μm in size) per container and ≤600 particles (≥25 μm insize) per container after storage at about 22° C. to about 28° C. for atleast about one month, at least about two months, at least about threemonths, at least about four months, at least about five months, at leastabout six months, at least about seven months, at least about eightmonths, at least about nine months, at least about ten months, at leastabout eleven months, or at least about twelve months.

The methods of the present invention produce stable therapeutic proteinpharmaceutical formulations resistance to sub-visible particulateformation during cold storage. In one embodiment, the methods produceformulations containing ≤6000 particles (≥10 μm in size) per containerand ≤600 particles (≥25 μm in size) per container after storage at about1° C. to about 10° C. for about six months, at least about 12 months, atleast about 18 months, at least about 24 months, at least about 30months, at least about 36 months, at least about 42 months, or at leastabout 48 months.

In another embodiment, the methods of the present invention producestable therapeutic protein pharmaceutical formulations resistance tosub-visible particulate formation during long-term cold storage. In oneembodiment, the methods described herein produce formulations containing≤6000 particles (≥10 μm in size) per container and ≤600 particles (≥25μm in size) per container after storage at about −15° C. to about −90°C. for at least about 12 months, at least about 18 months, at leastabout 24 months, at least about 30 months, at least about 36 months, atleast about 42 months, at least about 48 months, at least about 54months, at least about 60 months, at least about 66 months, or at leastabout 72 months.

In another embodiment, the methods of the present invention producestable formulations resistant to visible particulate formation. In oneembodiment, the methods produce formulations containing ≤80 particles(≥70 μm in size) per milliliter after storage at about 22° C. to about28° C. for at least about one month, at least about two months, at leastabout three months, at least about four months, at least about fivemonths, at least about six months, at least about seven months, at leastabout eight months, at least about nine months, at least about tenmonths, at least about eleven months, or at least about twelve months.In another embodiment, the methods described herein produce formulationscontaining ≤10 particles (≥70 μm in size) per milliliter after storageat about 22° C. to about 28° C. for at least about one month, at leastabout two months, at least about three months, at least about fourmonths, at least about five months, at least about six months, at leastabout seven months, at least about eight months, at least about ninemonths, at least about ten months, at least about eleven months, or atleast about twelve months.

The methods of the present invention produce stable therapeutic proteinpharmaceutical formulations resistance to visible particulate formationduring cold storage. In one embodiment, the methods described hereinproduce formulations containing ≤80 particles (≥70 μm in size) permilliliter after storage at about 1° C. to about 10° C. for about sixmonths, at least about 12 months, at least about 18 months, at leastabout 24 months, at least about 30 months, at least about 36 months, atleast about 42 months, or at least about 48 months. In anotherembodiment, the methods described herein produce formulations containing≤10 particles (≥70 μm in size) per milliliter after storage at about 1°C. to about 10° C. for at least about six months, at least about 12months, at least about 18 months, at least about 24 months, at leastabout 30 months, at least about 36 months, at least about 42 months, orat least about 48 months.

In another embodiment, the methods of the present invention producestable therapeutic protein pharmaceutical formulations resistance tovisible particulate formation during long-term cold storage. In oneembodiment, the methods described herein produce formulations containing≤80 particles (≥70 μm in size) per milliliter after storage at about−15° C. to about −90° C. for at least about 12 months, at least about 18months, at least about 24 months, at least about 30 months, at leastabout 36 months, at least about 42 months, at least about 48 months, atleast about 54 months, at least about 60 months, at least about 66months, or at least about 72 months. In another embodiment, the methodsdescribed herein produce formulations containing ≤10 particles (≥70 μmin size) per milliliter after storage at about −15° C. to about −90° C.for at least about 12 months, at least about 18 months, at least about24 months, at least about 30 months, at least about 36 months, at leastabout 42 months, at least about 48 months, at least about 54 months, atleast about 60 months, at least about 66 months, or at least about 72months.

In another embodiment, the methods of the present invention producestable formulations with low turbidity. In one embodiment, the methodsdescribed herein produce formulations having ≤18.0 NTU (NephelometricTurbidity Unit) after storage at about 22° C. to about 28° C. for atleast about one month, at least about two months, at least about threemonths, at least about four months, at least about five months, at leastabout six months, at least about seven months, at least about eightmonths, at least about nine months, at least about ten months, at leastabout eleven months, or at least about twelve months.

The methods of the present invention produce stable therapeutic proteinpharmaceutical formulations with low turbidity during cold storage. Inone embodiment, the methods described herein produce formulations having≤18.0 NTU after storage at about 1° C. to about 10° C. for about sixmonths, at least about 12 months, at least about 18 months, at leastabout 24 months, at least about 30 months, at least about 36 months, atleast about 42 months, or at least about 48 months.

In another embodiment, the methods of the present invention producestable therapeutic protein pharmaceutical formulations having lowturbidity during cold storage. In one embodiment, the turbidity of theformulations as described herein is ≤18.0 NTU after storage at about−15° C. to about −90° C. for at least about 12 months, at least about 18months, at least about 24 months, at least about 30 months, at leastabout 36 months, at least about 42 months, at least about 48 months, atleast about 54 months, at least about 60 months, at least about 66months, or at least about 72 months.

In another embodiment, the methods of the present invention producepharmaceutical formulations where the shelf life of the formulation isextended at least about one month, at least about two months, at leastabout three months, at least about four months, at least about fivemonths, at least about six months, at least about seven months, at leastabout eight months, at least about nine months, at least about tenmonths, at least about eleven months, or at least about twelve monthsbeyond the shelf life of a corresponding formulation containing apolysorbate surfactant.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and no way limit the scope of the invention.

EXAMPLES Materials and Methods

Materials: Surfactants investigated in this study included PFA andpolysorbates. Two PFA, Brij® 35 (L23) (Product #ET47399, CAS #9002-92-0)and Brij® 98 (O20) (Product #ET40118, CAS #9004-98-2), were obtainedfrom Croda International PLC (East Yorkshire, UK). Polysorbate 20(Product #4116-04, CAS #9005-64-5) and polysorbate 80 (Product #4117-03,CAS #9005-65-6) were obtained from JT Baker (Center Valley, Pa.). Lipaseimmobilized on polystyrene beads at 2.0 U/mg (Product Number 73940) wasobtained from Fluka (Denmark). All monoclonal antibodies (mAbs) wereproduced or acquired by Janssen Supply Chain, LLC (Horsham, Pa.) andpurified in a series of chromatography and filtration steps.

Size Exclusion Chromatography (SEC)

The soluble aggregation quality attribute of the investigatedformulations was evaluated using size exclusion ultra performance liquidchromatography (SE-UPLC). To prepare samples for analysis, collectedformulations from each condition (time zero (T0) control, shakingstress, and thermal stress followed by shaking stress) were diluted withformulation buffer of each respective mAb from 50 mg/mL to 2 mg/mL. SECruns were conducted with a 5 μL injection of sample into an Acquity UPLC200Å, 1.7 μm column using a 0.2 M sodium phosphate mobile phase. Elutingpeaks were integrated to determine monomer content based on percent areaof the main peak relative to sum of all peak areas.

Dynamic Light Scattering (DLS)

Particulate formation was assessed using dynamic light scattering. Toprepare samples for analysis, collected formulations from each condition(T0 control, shaking stress, and thermal stress followed by shakingstress) were diluted with formulation buffer of each respective mAb from50 mg/mL to 2 mg/mL. Diluted samples were then measured using a DLSinstrument at constant 20° C., with percent scattering intensityreported as a function of particle size range. Particle size binsincluded: 2-8 nm, 8-60 nm, 60-300 nm, and 300-10,000 nm.

Surfactant Quantitation

Surfactant quantitation of samples was performed using UPLC withevaporative light scattering detection. Duplicate 7.5 μL sampleinjections were injected into an OASIS MAX column (30 μm, 2.1×20 mm).The mobile phase consisted of a gradient of milliQ water and isopropanolwith 2% formic acid. The integrated area for each sample's eluting peakwas assessed against a surfactant standard curve created from serialdilution of each surfactant type ranging from 0.005% (w/v) to 0.08%(w/v).

Assay for Esterase/Lipase Activity

The enzymatically catalyzed hydrolysis of 4-Nitrophenyl acetate (Sigma,Product No.: N8130) was used as a model system to measureesterase/lipase activity (Valkova, N., et al., “Purification andcharacterization of PrbA, a new esterase from Enterobactercloacae</I>hydrolyzing the esters of 4-hydroxybenzoic acid (Parabens)”,J. Biol. Chem. 278(15), pp.12779-12785 (2003), John, G. T., and Heinzle,E., “Quantitative screening method for hydrolases in microplates usingpH indicators: determination of kinetic parameters by dynamic pHmonitoring”, Biotechnol. Bioeng. 72(6), pp.620-627 (2001), which arehereby incorporated by reference in their entirety). In brief, the assaymeasures the conversion of 4-Nitrophenyl Acetate to 4-Nitrophenol byesterase/lipase. At pH>6, the 4-Nitrophenol is deprotonated (phenolate)and is colored yellow. The presence of phenolate is monitored at 400 nmand the intensity of the color is pH dependent from 6.0 to 8.0. Tocompensate for differences in formulation buffer pH, 25 mM HEPES bufferat pH=7.5 was used to adjust the pH to 7.0 prior to the assay. Controlsfor the assay included, NaOH, HCl, porcine esterase (Sigma Product No.:E2884-5KU), water, and formulation buffer without protein.

Example 1—PFA Surfactants Protect mAbs from Shaking and ThermalStability Stresses

To assess the protective effects of different surfactants in mAbformulations, polysorbate and PFA was spiked into formulations for threedifferent mAbs. mAbs A and B are bispecific antibodies, and mAb C is anIgG4 monoclonal antibody. The formulations for mAbs A and B comprised 10mM histidine, 8.5% (w/v) sucrose, at pH 5.7. The mAb C formulationconsisted of 10 mM sodium phosphate, 8.5% (w/v) sucrose, 10 ppm EDTA, atpH 7.1. All three mAbs were at a 50 mg/mL concentration in theirrespective formulations. Stock 3% (w/v) surfactant solutions were spikedinto the mAb formulations to yield 0.01% and 0.04% (w/v) polysorbate orPFA concentrations in each mAb's final formulation. After eachformulation was prepared, the material was filled into autoclaved 2Rglass vials with a 1 mL fill, stoppered, and crimp sealed. These vialedformulations were then divided into three conditions: T0 control,shaking stress (72 hours on an orbital shaker at 250 RPM in ambienttemperature), and thermal stress (3-month incubation at 25° C.) followedby shaking stress (72 h at 250 RPM). Each formulation condition wasevaluated with vials in triplicate. The impact of the surfactant typeand concentration was assessed using size exclusion chromatography(SEC).

FIGS. 1A-1C are graphs showing the percentage of each formulation thatis in the desired monomeric state as assessed by SEC. The comparison ofpolysorbate and PFA containing formulations reveals similar percentmonomer results when formulations are exposed to shaking and thermalstresses. At the low 0.01% (w/v) surfactant level, SEC results indicatethat PFA (Brij O20 and Brij L23) formulations retain higher percentmonomer content than polysorbate (PS20 and PS80) formulations for thestressed condition of 3-month storage at 25° C. following by shakingstress (Student's t-test, p<0.01). These findings suggest PFAformulations may be able to provide more protection against thermal andshaking stresses than polysorbate at low concentrations.

Example 2—PFA Sock Solutions Exposed to Lipases Immobilized on BeadsExhibit Significant Resistance to Degradation.

Stock solutions of PFA and polysorbate surfactants were exposed tolipases immobilized on beads over a period of 18 days. Surfactant levelsin the solutions were quantified periodically over the course of testingto determine the extent of lipase-mediated degradation.

The quantitation of intact polysorbate levels in solutions exposed tolipases suggested a >50% reduction of intact polysorbate content after 3days (see FIG. 2). In contrast, the PFA surfactant levels remainedessentially unchanged for the entire duration of exposure, up to 18 days(FIG. 2). These results highlight the difference in resistance tolipase-mediated degradation of PFA versus polysorbate, and demonstratethat PFA surfactants would remain intact in mAb formulations despiteresidual host cell proteins from the antibody manufacturing process.

Example 3—mAb Stability is Maintained in Formulations Containing PFASurfactant Exposed to Lipase

Further studies evaluated the ability of PFA and polysorbateformulations to retain their protective effects after exposures of thesurfactants to lipase. The mAb formulations were spiked with surfactantsthat had been exposed to lipase-beads and then subjected to shakingstress or thermal stress followed by shaking. The mAb formulations werethen evaluated for soluble aggregation and particulate matter using SECand DLS, respectively.

The SEC results demonstrate the deleterious effects of lipase exposureon polysorbate surfactants and the associated impact on mAb stability.In some cases, the presence of polysorbate exposed to lipase resulted inlower percent monomer content than control formulations without anysurfactant (see FIG. 3A, compare 0.01% PS80 and 0.04% PS80 to NoSurfactant, and FIG. 3C, compare 0.01% PS20 to No Surfactant). Thishighlights the disadvantage of using polysorbate surfactants informulation circumstances involving lipase exposure. By contrast, acrosseach investigated mAb, the PFA formulations retained their protectiveeffects despite lipase exposure. At 0.01% (w/v) surfactant level, PFAformulations of each mAb outperform polysorbate formulations and nosurfactant controls for all stress conditions. At the higher 0.04% (w/v)surfactant level, certain mAbs do not exhibit a strong decrease insoluble aggregate as measured by SEC, but the detrimental effect oflipase-mediated polysorbate degradation is instead evident by insolubleparticulate formation.

The impact of lipase exposure on formulation stability was alsodemonstrated in orthogonal assays such as DLS which evaluate particulateformation as a function of surfactant. The DLS particle sizedistribution results show that polysorbate formulations exposed tolipases are more prone to larger size particulate formation than PFAformulations (see FIGS. 4A-4C). For both low and high surfactant level,essentially all the scattering intensity in PFA formulations derivesfrom particle sizes binned in peak 1, which correlates to a monomer sizerange. This contrasts to the polysorbate formulations, which have asignificant amount of scattering derived from larger particulatesranging from 8 nm to 300 nm (FIGS. 4A-4B).

The orthogonal DLS assay complements SEC results and demonstrates thatPFA formulations outperform polysorbate formulations in their ability toprotect the mAb from soluble aggregation and insoluble particulateformation in conditions where lipases can degrade ester bond containingsurfactants.

Example 4—An Esterase/Lipase Activity Level ≥1 Units/mL is Detrimentalto Therapeutic Protein Formulations

To determine the threshold level of host cell derived esterase/lipaseactivity that is detrimental to a therapeutic protein formulation,esterase/lipase activity was determined in formulations containingdifferent concentrations of purified therapeutic proteins (antibodies).A representative example is shown in FIG. 5, with analysis ofesterase/lipase activity in mAb D formulations with concentrations of 90mg/ml and 5 mg/ml. As shown in FIG. 5, mAb D formulations containing 5mg/mL total protein exhibited a lower level of esterase/lipase activity(see last bar in graph of FIG. 5). While this level of esterase/lipaseactivity was detectable and the effects measurable, it was determinedthat esterase/lipase activity below this level of activity could beacceptable with regard to polysorbate degradation and stability of atherapeutic protein. Thus, the esterase/lipase activity below what ispresent in the 5 mg/mL sample of mAb D was determined to be anacceptable esterase/lipase activity level. A more prudent cutoff for anacceptable level of esterase/lipase activity would be activity that is<10% of the activity present in the 5 mg/mL sample of mAb D.

Data analysis determined that the esterase/lipase activity in the 5mg/mL mAb D sample represented 1% of the esterase control (porcineesterase; Sigma PN E2884-5KU, CAS#9016-18-6, EC3.1.1.1) having 125 U/mLactivity. Based on that analysis, an acceptable level of esterase/lipaseactivity in the mAb D formulation corresponds to <1.25 Units/mL ofporcine esterase. Rounding to the correct number of significant figures,an acceptable level of esterase/lipase activity in the mAb D formulationcorresponds to <1 Units/mL of porcine esterase. In certain embodiments,an acceptable level of esterase/lipase activity in the mAb D formulationcorresponds to <1, <0.9, <0.8, <0.7, <0.6, <0.5, <0.4, <0.3, <0.2, or<0.1 Units/mL of porcine esterase. In certain embodiments, an acceptablelevel of esterase/lipase activity in the mAb D formulation correspondsto <0.1 Units/mL of porcine esterase. Thus, an esterase/lipase activitylevel corresponding to ≥1 Units/mL of porcine esterase is considered tobe detrimental to a therapeutic protein formulation. In certain otherembodiments, esterase/lipase activity level corresponding to ≥1, ≥0.9,≥0.8, ≥0.7, ≥0.6, ≥0.5, ≥0.4, ≥0.3, ≥0.2, or ≥0.1 Units/mL of porcineesterase is considered to be detrimental to a therapeutic proteinformulation. In certain other embodiments, esterase/lipase activitylevel corresponding to ≥0.1 Units/mL of porcine esterase is consideredto be detrimental to a therapeutic protein formulation. According to thedata presented herein, a polysorbate surfactant may not be the bestchoice for a formulation with an esterase/lipase activity levelcorresponding to ≥1 Units/mL of porcine esterase. In certain otherembodiments, a polysorbate surfactant may not be the best choice for aformulation with an esterase/lipase activity level corresponding to ≥1,≥0.9, ≥0.8, ≥0.7, ≥0.6, ≥0.5, ≥0.4, ≥0.3, ≥0.2, or ≥0.1 Units/mL ofporcine. Rather, the data suggests that polysorbate surfactants shouldbe avoided in formulations with an esterase/lipase activity levelcorresponding to ≥1 Units/mL of porcine esterase. In addition, the datasuggests that polysorbate surfactants should be avoided in formulationswith an esterase/lipase activity level corresponding to ≥1, ≥0.9, ≥0.8,≥0.7, ≥0.6, ≥0.5, ≥0.4, ≥0.3, ≥0.2, or ≥0.1 Units/mL of porcineesterase. In certain other embodiments, polysorbate surfactants shouldbe avoided in formulations with an esterase/lipase activity levelcorresponding to ≥0.1 Units/mL of porcine esterase. Instead, apolyethoxylated fatty alcohol surfactant as described herein should beused in a formulation with an esterase/lipase activity levelcorresponding to ≥1 Units/mL of porcine esterase. In certain otherembodiments, a polyethoxylated fatty alcohol surfactant as describedherein should be used in a formulation with an esterase/lipase activitylevel corresponding to ≥1, ≥0.9, ≥0.8, ≥0.7, ≥0.6, ≥0.5, ≥0.4, ≥0.3,≥0.2, or ≥0.1 Units/mL of porcine esterase. In certain otherembodiments, a polyethoxylated fatty alcohol surfactant as describedherein should be used in a formulation with an esterase/lipase activitylevel corresponding to ≥0.1 Units/mL of porcine esterase.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

What is claimed is:
 1. A pharmaceutical formulation comprising: about 20mg/mL to about 200 mg/mL of a therapeutic protein; a pharmaceuticallyacceptable carrier; and one or more polyethoxylated fatty alcohol (PFA)surfactants, wherein said one or more PFA surfactants is resistant tolipase degradation.
 2. The pharmaceutical formulation of claim 1,wherein the therapeutic protein is a non-recombinant, serum isolatedprotein, a recombinant protein, an antibody or an antigen bindingportion thereof, or an antibody-drug conjugate.
 3. The method of claim1, wherein the concentration of the therapeutic protein in theformulation is about 50 mg/mL to about 150 mg/mL
 4. The pharmaceuticalformulation of claim 1, wherein the one or more PFA surfactants comprisea PFA having between 5 to 40 ethylene glycol units.
 5. Thepharmaceutical formulation of claim 4, wherein the one or more PFAsurfactants is polyoxyethylene (23) lauryl ether and/or polyoxyethylene(20) oleyl ether.
 6. The pharmaceutical formulation of claim 1, whereinthe PFA surfactant concentration in the formulation is about 0.005% toabout 0.2% (w/v).
 7. The pharmaceutical formulation of claim 1, whereinthe formulation comprises lipase/esterase activity that is ≥1 unit/mL ofpurified porcine esterase or equivalent thereof.
 8. The pharmaceuticalformulation of claim 1, wherein >90% the PFA surfactant remains intactin the formulation over the formulation's shelf life.
 9. Thepharmaceutical formulation of claim 1, wherein the therapeutic proteinwas produced in a cell line selected from the group consisting ofChinese hamster ovary (CHO) cell line, PER.C6 cell line, and Sp2/0 cellline.
 10. The pharmaceutical formulation of claim 1 further comprising:a saccharide, said saccharide comprising about 0.5% to 15% w/v of theformulation; and, buffering salts in a concentration of about 5 mM to 50mM.
 11. The pharmaceutical formulation of claim 1, wherein saidformulation has a pH of between 5-8.
 12. The pharmaceutical formulationof claim 1, wherein the formulation is resistant to particulateformation.
 13. The pharmaceutical formulation of claim 12, wherein theformulation contains ≤80 particle/mL of particles having an equivalentcircular diameter of ≥70 μm over formulation's shelf life.
 14. Thepharmaceutical formulation of claim 12, wherein the formulation contains≤6000 particles (≥10 μm in size) per container and ≤600 particles (≥25μm in size) per container over the formulation's shelf life.
 15. Thepharmaceutical formulation of claim 1, wherein the formulation isresistant to protein aggregation.
 16. The pharmaceutical formulation ofclaim 15, wherein >90% of the protein in the formulation is in anon-aggregated state over the formulation's shelf life.
 17. A method ofreducing aggregate and/or particulate formation in a pharmaceuticalformulation comprising a biological composition, said method comprising:providing a biological composition, said composition comprising about 20mg/mL to about 200 mg/mL of therapeutic protein and incorporating one ormore lipase resistant polyethoxylated fatty alcohol (PFA) surfactants inthe biological composition as a replacement for a polysorbatesurfactant.
 18. The method of claim 17, wherein the therapeutic proteinof the biological composition is a non-recombinant, serum isolatedprotein, a recombinant protein, an antibody or an antigen bindingportion thereof, or an antibody-drug conjugate.
 19. The method of claim17, wherein the concentration of the therapeutic protein in thebiological composition is about 50 mg/mL to about 150 mg/mL
 20. Themethod of claim 17, wherein the one or more PFA surfactants comprise aPFA having between 5 to 40 ethylene glycol units.
 21. The method ofclaim 17, wherein the one or more PFA surfactants is polyoxyethylene(23) lauryl ether and/or polyoxyethylene (20) oleyl ether.
 22. Themethod of claim 17, wherein the PFA surfactant is incorporated into thepharmaceutical formulation in an amount of about 0.005% to about 0.2%(w/v).
 23. The method of claim 17, wherein the therapeutic protein wasproduced in a cell line selected from the group consisting of CHO cellline, PER.C6 cell line, and Sp2/0 cell line.
 24. The method of claim 17further comprising: measuring lipase activity of the biologicalcomposition, wherein said incorporating is based on said measuring. 25.The method of any of claims 17-24, wherein said biological compositioncomprises a lipase/esterase activity that is ≥1 unit/mL of purifiedporcine esterase or equivalent thereof.
 26. The method of any of claims17-24, wherein said biological composition comprises a lipase/esteraseactivity that is ≥0.1 unit/mL of purified porcine esterase or equivalentthereof.